Arginine Analogues Research Articles

A Proteasomal ATPase Contributes to Dislocation of Endoplasmic Reticulum-associated Degradation (ERAD) Substrates

Endoplasmic reticulum (ER)-associated degradation (ERAD) eliminates aberrant proteins from the ER by dislocating them to the cytoplasm where they are tagged by ubiquitin and degraded by the proteasome. Six distinct AAA-ATPases (Rpt1-6) at the base of the 19S regulatory particle of the 26S proteasome recognize, unfold, and translocate substrates into the 20S catalytic chamber. Here we show unique contributions of individual Rpts to ERAD by employing equivalent conservative substitutions of the invariant lysine in the ATP-binding motif of each Rpt subunit. ERAD of two substrates, luminal CPY*-HA and membrane 6myc-Hmg2, is inhibited only in rpt4R and rpt2RF mutants. Conversely, in vivo degradation of a cytosolic substrate, DeltassCPY*-GFP, as well as in vitro cleavage of Suc-LLVY-AMC are hardly affected in rpt4R mutant yet are inhibited in rpt2RF mutant. Together, we find that equivalent mutations in RPT4 and RPT2 result in different phenotypes. The Rpt4 mutation is manifested in ERAD defects, whereas the Rpt2 mutation is manifested downstream, in global proteasomal activity. Accordingly, rpt4R strain is particularly sensitive to ER stress and exhibits an activated unfolded protein response, whereas rpt2RF strain is sensitive to general stress. Further characterization of Rpt4 involvement in ERAD reveals that it participates in CPY*-HA dislocation, a function previously attributed to p97/Cdc48, another AAA-ATPase essential for ERAD of CPY*-HA but dispensable for proteasomal degradation of DeltassCPY*-GFP. Pointing to Cdc48 and Rpt4 overlapping functions, excess Cdc48 partially restores impaired ERAD in rpt4R, but not in rpt2RF. We discuss models for Cdc48 and Rpt4 cooperation in ERAD.

Some physicochemical and antinutritional properties of raw flours and protein isolates from Mucuna pruriens (velvet bean) and Canavalia ensiformis (jack bean)

SummaryThe legumes Canavalia ensiformis and Mucuna pruriens are underexploited in tropical Mexico. Their seeds have good nutritional potential, but contain antinutritional factors. Physicochemical and antinutritional properties were determined for raw flours (RF) and protein isolates (PI) produced from these legumes. Protein content in the PI was 737 g kg–1 for C. ensiformis and 666 g kg–1 for M. pruriens. Protein isolation improved in vitro digestibility, while maintaining high lysine levels and adequate sulphur amino acids content. Antinutritional factors such as cyanogenic glucosides, cyanide precursors from hydrolysis, tannins and trypsin inhibitors were lower in the PIs than in the RFs. The reduction in canavanine levels, a structural analogue of arginine, in the C. ensiformis PI was noteworthy. These PIs thus have potential applications in the development of new food ingredients in tropical regions using processes that improve nutritional value.

Inactivation of Microbial Arginine Deiminases by l-Canavanine

Arginine deiminase (ADI) catalyzes the hydrolytic conversion of L-arginine to ammonia and L-citrulline as part of the energy-producing L-arginine degradation pathway. The chemical mechanism for ADI catalysis involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate. The structure of the Pseudomonas aeruginosa ADI-(L-arginine) complex guided the design of arginine analogs that might react with the ADIs to form inactive covalent adducts during catalytic turnover. One such candidate is L-canavanine, in which an N-methylene of L-arginine is replaced by an N-O. This substance was shown to be a slow substrate-producing O-ureido-L-homoserine. An in depth kinetic and mass spectrometric analysis of P. aeruginosa ADI inhibition by L-canavanine showed that two competing pathways are followed that branch at the Cys-alkylthiouronium ion intermediate. One pathway leads to direct formation of O-ureido-L-homoserine via a reactive thiouronium intermediate. The other pathway leads to an inactive form of the enzyme, which was shown by chemical model and mass spectrometric studies to be a Cys-alkylisothiourea adduct. This adduct undergoes slow hydrolysis to form O-ureido-L-homoserine and regenerated enzyme. In contrast, kinetic and mass spectrometric investigations demonstrate that the Cys-alkylthiouronium ion intermediate formed in the reaction of L-canavanine with Bacillus cereus ADI partitions between the product forming pathway (O-ureido-L-homoserine and free enzyme) and an inactivation pathway that leads to a stable Cys-alkylthiocarbamate adduct. The ADIs from Escherichia coli, Burkholderia mallei, and Giardia intestinalis were examined in order to demonstrate the generality of the L-canavanine slow substrate inhibition and to distinguish the kinetic behavior that defines the irreversible inhibition observed with the B. cereus ADI from the time controlled inhibition observed with the P. aeruginosa, E. coli, B. mallei, and G. intestinalis ADIs.

Mechanisms of Vasculopathy in Sickle Cell Disease and Thalassemia

Many mechanisms contribute to the complex pathophysiology of sickle cell disease (SCD), with dysfunction of the vascular endothelium as a unifying theme. Specifically, hemolysis-associated low arginine and nitric oxide (NO) bioavailability, amplified by NO synthase uncoupling, elevated arginase activity, superoxide production, oxidative stress, accumulation of arginine analogs such as asymmetric dimethylarginine, ischemia-reperfusion injury, inflammation, apolipoprotein A-1 depletion, and a hypercoagulable state are significant mechanisms contributing to endothelial dysfunction. Genetic polymorphisms also influence disease severity. Clearly the variable spectrum of disease is the consequence of multiple events and genetic susceptibility that go beyond the occurrence of a single amino acid substitution in the beta globin chain of hemoglobin. Recent studies begin to demonstrate overlap among these seemingly unrelated processes. Impaired NO bioavailability represents the central feature of endothelial dysfunction, and is a common denominator in the pathogenesis of vasculopathy in SCD. The consequences of decreased NO bioavailability include endothelial cell activation, upregulation of the potent vasoconstrictor endothelin-1, vasoconstriction, platelet activation, increased tissue factor, and activation of coagulation, all of which ultimately translate into the clinical manifestations of SCD. Evidence supporting vasculopathy subphenotypes in SCD, including pulmonary hypertension, priapism, cutaneous leg ulceration, and stroke, will be reviewed and relevance to other hemolytic disorders including the thalassemia syndromes will be considered.

Role of nitric oxide in inflammatory diseases

Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation. It gives an anti-inflammatory effect under normal physiological conditions. On the other hand, NO is considered as a pro-inflammatory mediator that induces inflammation due to over production in abnormal situations. NO is synthesized and released into the endothelial cells by the help of NOSs that convert arginine into citrulline producing NO in the process. Oxygen and NADPH are necessary co-factors in such conversion. NO is believed to induce vasodilatation in cardiovascular system and furthermore, it involves in immune responses by cytokine-activated macrophages, which release NO in high concentrations. In addition, NO is a potent neurotransmitter at the neuron synapses and contributes to the regulation of apoptosis. NO is involved in the pathogenesis of inflammatory disorders of the joint, gut and lungs. Therefore, NO inhibitors represent important therapeutic advance in the management of inflammatory diseases. Selective NO biosynthesis inhibitors and synthetic arginine analogues are proved to be used for the treatment of NO-induced inflammation. Finally, the undesired effects of NO are due to its impaired production, including in short: vasoconstriction, inflammation and tissue damage.

Inhibition of Human Dimethylarginine Dimethylaminohydrolase-1 by S-Nitroso-L-homocysteine and Hydrogen Peroxide

The plasma concentrations of two cardiovascular risk factors, total homocysteine (tHcy) and asymmetric dimethylarginine (ADMA), correlate with decreased levels of endothelium-derived nitric oxide and subsequent endothelial dysfunction. Homocysteine has been proposed to inhibit the catabolic enzyme of ADMA, dimethylarginine dimethylaminohydrolase (DDAH), but the mechanism of this inhibition has not been fully elucidated. Here, the human DDAH isoform-1 (DDAH-1) is heterologously expressed and purified. Cys(274) and His(173) are identified as active site residues and the pH rate dependence is described. Because oxidation of the active site Cys has been suggested as an inhibitory mechanism in patients with hyperhomocysteinemia, the sensitivity of DDAH-1 to inhibition by L-homocysteine, H(2)O(2), and S-nitroso-L-homocysteine is quantified. DDAH-1 is surprisingly insensitive to inactivation by the powerful oxidant, H(2)O(2) (0.088 M(-1) s(-1)), possibly because of a substrate-assisted mechanism that allows the active site cysteine to remain predominantly protonated and less reactive in the resting enzyme. In contrast, DDAH-1 is sensitive to inactivation by S-nitroso-L-homocysteine (3.79 M(-1) s(-1)). This work illustrates how a particular catalytic mechanism can result in selective redox regulation and has possible implications for hyperhomocysteinemia.

Design and Synthesis of C5 Methylated l-Arginine Analogues as Active Site Probes for Nitric Oxide Synthase

The role of nitric oxide (NO) as a biological signaling molecule is well established. NO is produced by the nitric oxide synthases (NOSs, EC 1.14.13.39), a class of heme proteins capable of converting l-arginine to NO and l-citrulline. Despite the large body of knowledge associated with the NOSs, mechanistic details relating to the unique oxidative chemistry performed by these enzymes remain to be fully elucidated. Furthermore, a number of disease states are associated with either the over- or underproduction of NO, making the NOS pathway an attractive target for the development of therapeutics. For these reasons, molecular tools capable of providing mechanistic insights into the production of NO and/or the inhibition of the NOSs remain of interest. We report here the stereospecific synthesis and testing of a number of new l-arginine analogues bearing a minimal substitution, methylation at position 5 of the amino acid side chain (such analogues have not been previously reported). The synthetic approach employed a modified photolysis procedure whereby irradiation of the appropriate diacylperoxide precursors at 254 nm gave access to the required unnatural amino acids in good yields. A heme domain construct of the inducible NOS isoform (iNOSheme) was used to assess the binding of each compound to the enzyme active site. The compounds were also investigated as either inhibitors of, or alternate substrates for, the inducible NOS isoform. The results obtained provide new insight into the steric and stereochemical tolerance of the enzyme active site. These findings also further support the role of a conserved active site water molecule previously proposed to be necessary for NOS catalysis.

Chemosensory reception, behavioral expression, and ecological interactions at multiple trophic levels

Chemoreception may function throughout an entire animal lifetime, with independent, stage-specific selection pressures leading to changes in physiological properties, behavioral expression, and hence, trophic interactions. When the California newt (Taricha torosa) metamorphoses from an entirely aquatic larva to a semi-terrestrial juvenile/adult form, its chemosensory organs undergo dramatic reorganization. The relationship between newt life-history stage and chemosensory-mediated behavior was established by comparing responses of adults (as determined here) to those of conspecific larvae (as studied previously). Bioassays were performed in mountain streams, testing responses of free-ranging adults to 13 individual l-amino acids. Relative to stream water (controls), adults turned immediately upcurrent and moved to the source of arginine, glycine or alanine release. These responses were indicative of predatory search. Arginine was the strongest attractant tested, with a response threshold (median effective dose) of 8.3x10(-7) mol l(-1) (uncorrected for dilution associated with chemical release and delivery). In contrast to adult behavior, arginine suppressed cannibal-avoidance and failed to evoke search reactions in larvae. For a common set of arginine analogs, the magnitudes of adult attraction and larval suppression were not positively correlated. Suppression of cannibal-avoidance behavior in larvae was unaffected by most structural modifications of the arginine molecule. Adult behavior, on the other hand, was strongly influenced by even subtle alterations in the parent compound. Reactions to arginine in both adults and larvae were eliminated by blocking the external openings of the nasal cavity. Stimulating adult predatory search in one case and inhibiting larval cannibal avoidance in the other, arginine is a chemical signal with opposing behavioral effects and varying ecological consequences. Significant differences between responses of adults and larvae to changes in arginine structure suggest alternative, chemosensory receptor targets. Although arginine reception functions throughout an entire newt lifetime, an ontogenetic shift in larval and adult chemoreceptive ability changes behavioral expression, and thus, reflects the unique selection pressures that act at each life-history stage.

Synthesis of Proteins Containing Modified Arginine Residues

Unnatural amino acid mutagenesis provides the wherewithal to study protein function in great detail. To extend the repertoire of functionalized amino acids available for study by this technique, seven structural analogues of arginine were prepared and used to activate a suppressor tRNACUA. These included Ngamma-methylarginine, Ngamma-nitroarginine, citrulline, homoarginine, and three conformationally constrained analogues based on proline. These misacylated tRNAs were shown to be capable of introducing the arginine analogues into dihydrofolate reductase (position 22) and Photinus pyralis luciferase (positions 218 and 437). Most of the modified luciferases containing arginine analogues at position 218 emitted light with less efficiency and at longer wavelength than the wild type. This is consistent with the postulated role of this residue as essential for maintaining the polarity and rigidity of the luciferin-binding site. Interestingly, the luciferase containing Ngamma-methylarginine at position 218 emitted light at the same wavelength as the wild type and was at least as efficient. Alteration of the arginine residue at position 437 had no effect on the wavelength of emitted light but afforded analogues, all of which emitted light less efficiently than the wild type. This is altogether consistent with the putative role of Arg437, which is an invariant residue within the superfamily of enzymes that includes P. pyralis luciferase. This amino acid is part of the linker between the two structural domains of luciferase that is believed to be essential for efficient enzyme function but not part of the substrate-binding site.

THE RESPONSE OF MRL/LPR-INDUCIBLE NITRIC OXIDE SYNTHASE KNOCKOUT MICE TO INDUCIBLE NITRIC OXIDE SYNTHASE INHIBITOR.

Increases in systemic nitric oxide (NO) production are associated with disease progression, and pharmacologic inhibition of inducible nitric oxide synthase (iNOS) abrogates disease in the MRL/lpr murine model of lupus. However, genetic manipulation of iNOS alone does not improve inflammatory glomerular or joint disease in these mice. We hypothesize that the arginine analog iNOS inhibitors used either had non-iNOS-mediated effects or they inhibit potentially pathogenic activity in other NOS isoforms as well. To address these hypotheses, 21 mice (either iNOS −/− mutants or iNOS+/+ littermates) on the MRL/lpr background were divided into three groups: (1) seven iNOS+/+ littermates given placebo, (2) seven iNOS−/− mice given placebo, and (3) seven iNOS−/− mice given an iNOS inhibitor (SC-51) by subcutaneous sustained release pellet, given between ages 10 and 24 weeks of age. Urine nitrate + nitrite (NOX in μmol/mouse/d) and albumin (μg/mouse/d) levels were measured biweekly. Mice were sacrificed at 24 weeks, and the kidneys were sectioned, stained, and scored for glomerular pathology by light microscopy. NOX levels were greatest among the wild-type littermates (1.7 ± 1.5) than either of the knockout groups (p

Identification of Arginine Analogues as Antagonists and Agonists for the Melanocortin-4 Receptor

In the present study, conducted to explore potent and small molecular melanocortin-4 (MC4) receptor ligands, we found that tripeptide 3a, containing a D-Phe-Arg-2-Nal (Nal; naphthylalanine) sequence, exhibited a moderate affinity for the MC4 receptor. Structural optimization led to the identification of a compound with a high affinity for the MC4 receptor, namely, tripeptide 3e, which showed a 70-fold higher affinity for the MC4 receptor than the lead compound 3a. Moreover, in an effort to further reduce the peptidic characters of tripeptide 3e, we found that dipeptide 3g exhibited a relatively high affinity for the MC4 receptor. Furthermore, in these analogues, the substituted position (1' vs. 2') of the naphthyl ring of Nal residue at position 7 was found to be important for the differentiation of agonist and antagonist activity. The synthesis and structure-activity relationships of the arginine analogues as MC4 receptor ligands were described in this paper.

The lung in the balance: arginine, methylated arginines, and nitric oxide

the lung is a major source of nitric oxide (NO), be it from nitric oxide synthase (NOS) III in the endothelium of the vast pulmonary circulation, NOS II in the epithelium of the large surface area of the airways, or NOS I in the nonadrenergic noncholinergic nerves ([6][1]). NO produced in the lung

Dimethylarginine dimethylaminohydrolase (DDAH) regulates trophoblast invasion and motility through effects on nitric oxide

Invasion of trophoblast into the uterine environment is crucial for establishing a successful pregnancy. Physiological production of nitric oxide (NO) by extravillous trophoblasts results in significant pro-invasive effects. NO synthesis is competitively inhibited by methylated arginine analogues such as asymmetric dimethylarginine (ADMA) but not the enantiomer symmetric dimethylarginine (SDMA). Within cells, the concentration of ADMA is regulated by the activity of the enzyme dimethylarginine dimethylaminohydrolase (DDAH). The aim of this study was to examine DDAH expression and function in trophoblasts. DDAH-1 and DDAH-2 messenger RNA and protein were demonstrated in first trimester placental tissue, primary extravillous trophoblasts and extravillous trophoblast-derived cell lines. DDAH activity was demonstrated in both cells and tissue. Overexpression of DDAH-1 in trophoblasts led to a number of significant changes, including an 8-fold increase in enzymatic activity, a 59% decrease in production of ADMA (but not SDMA), a 1.9-fold increase in NO and a 1.6-fold increase in cyclic guanosine monophosphate (cGMP) production. Functional assays showed that increased DDAH activity led to significantly increased cell motility and invasion in response to hepatocyte growth factor (HGF). DDAH may play an important role in the regulation of extravillous trophoblast function via its effects on ADMA and NO production.

Vascular Action of Cocoa Flavanols in Humans: The Roots of the Story

Diet patterns are widely recognized as contributors to hypertension. Widely studied potential contributors include intake of sodium, potassium, magnesium, calcium, soluble fiber, omega-3 fatty acids, alcohol, protein, and calories. We add to that list the effect of dietary flavanols present in certain cocoas, which have sufficient activity on vascular nitric oxide to influence blood pressure control. Kuna Indians who live on islands near Panama have little age-related rise in blood pressure or hypertension. On migration to Panama City, blood pressure rises with age, and the frequency of essential hypertension matches urban levels elsewhere. We have identified a specific food that probably makes an important contribution to cardiovascular status. Island-dwelling Kuna drink more than 5 cups of flavanol-rich cocoa per day and incorporate that cocoa into many recipes. Mainland Kuna ingest little cocoa, and what they take is commercially available and flavanol-poor. The flavanol-rich cocoa activates nitric oxide synthase in vitro and in intact humans in the doses that the Kuna employ. Vasodilator responses to flavonoid-rich cocoa are prevented or reversed by the arginine analog, N-nitro-L-arginine methyl ester. Island-dwelling Kuna have a 3-fold larger urinary nitrate:nitrite than do Mainland dwellers. As endothelial dysfunction is central to current thinking on cardiovascular pathophysiology, a food that enhances endothelial function could have broad implications. The list of candidate conditions that might be influenced is impressive, ranging from atherosclerosis and diabetes mellitus to hypertension and preeclampsia, to vascular dementias and end-stage renal disease. The next decade will be interesting.

Cocoa Flavanols and Brain Perfusion

Foods and beverages rich in flavonoids are being heralded as potential preventive agents for a range of pathologic conditions, ranging from hypertension to coronary heart disease to stroke and dementia. We and others have demonstrated that short-term ingestion of cocoa, particularly rich in the subclass of flavonoids known as flavanols, induced a consistent and striking peripheral vasodilation in healthy people, improving endothelial function in a nitric oxide-dependent manner. The vasodilator response was reversed by N-nitro-L-arginine methyl ester, an arginine analog that blocks nitric oxide synthesis. Flavanol-poor cocoa induced much smaller responses. Because impairment of endothelial function is a nearly universal accompaniment of the aging process, we examined the peripheral vasodilator response to flavanol-rich cocoa in healthy older subjects. Observations point to a favorable response among the older. Together with peripheral vascular disease, cerebrovascular disease is responsible for significant mortality with advancing age. An association of decreased cerebral perfusion with dementia has been recently highlighted. The prospect of increasing cerebral perfusion with cocoa flavanols is extremely promising. Our still preliminary data hold out the promise that the cerebral blood supply in the elderly participates in the vasodilator response. With the modalities of transcranial Doppler and MRI, we have the capabilities of analyzing the potential benefits of flavanols on brain perfusion and, subsequently, on cognition.

Arginine, arginine analogs and nitric oxide production in chronic kidney disease

Nitric oxide (NO) production is reduced in renal disease, partially due to decreased endothelial NO production. Evidence indicates that NO deficiency contributes to cardiovascular events and progression of kidney damage. Two possible causes of NO deficiency are substrate (L-arginine) limitation and increased levels of circulating endogenous inhibitors of NO synthase (particularly asymmetric dimethylarginine [ADMA]). Decreased L-arginine availability in chronic kidney disease (CKD) is due to perturbed renal biosynthesis of this amino acid. In addition, inhibition of transport of L-arginine into endothelial cells and shunting of L-arginine into other metabolic pathways (e.g. those involving arginase) might also decrease availability. Elevated plasma and tissue levels of ADMA in CKD are functions of both reduced renal excretion and reduced catabolism by dimethylarginine dimethylaminohydrolase (DDAH). The latter might be associated with loss-of-function polymorphisms of a DDAH gene, functional inhibition of the enzyme by oxidative stress in CKD and end-stage renal disease, or both. These findings provide the rationale for novel therapies, including supplementation of dietary L-arginine or its precursor L-citrulline, inhibition of non-NO-producing pathways of L-arginine utilization, or both. Because an increase in ADMA has emerged as a major independent risk factor in end-stage renal disease (and probably also in CKD), lowering ADMA concentration is a major therapeutic goal; interventions that enhance the activity of the ADMA-hydrolyzing enzyme DDAH are under investigation.

Known regulators of nitric oxide synthase and arginase are agonists at the human G‐protein‐coupled receptor GPRC6A

GPRC6A is a novel family C G-protein-coupled receptor (GPCR) with so far unknown physiological function. It was the aim of our study to further characterize the ligand preferences of the receptor and elucidate structural requirements for activity. We have previously generated a functional chimeric receptor construct, h6A/5.24, containing the ligand-binding amino-terminal domain of the human GPRC6A and the seven-transmembrane domain and carboxy terminus of the homologous goldfish receptor 5.24. Based on knowledge that this chimera prefers basic L-alpha-amino acids such as arginine, lysine and ornithine, we searched for commercially available analogues of these and other L-alpha-amino acids, and tested them for activity in a fluorescence-based calcium assay. The majority of the tested compounds are involved in the regulation of nitric oxide synthase (NOS) and arginase enzymes. Altogether we profiled 30 different analogues. We found that a structurally wide range of L-alpha-amino-acid analogues of both arginine, lysine, and ornithine are agonists at h6A/5.24, whereas no antagonists were identified. From the profiling it is concluded that L-alpha-amino acids containing a highly basic side chain confer the highest activity, although the most potent compound was only twice as potent as L-arginine, which has a EC50 value of 23.5 microM. The reported agonism of NOS- and arginase-active compounds at GPRC6A has obvious implications in interpretation of experiments involving the NOS and arginase systems, and interfering effects at GPRC6A should be regarded of relevance, especially as the physiological function of the receptor is not yet understood.

RNA polymerase II subunit Rpb9 is important for transcriptional fidelity in vivo

The fidelity of yeast RNA polymerase II (Pol II) was assessed in vivo with an assay in which errors in transcription of can1-100, a nonsense allele of CAN1, result in enhanced sensitivity to the toxic arginine analog canavanine. The Pol II accessory factor TFIIS has been proposed to play a role in transcript editing by stimulating the intrinsic nuclease activity of the RNA polymerase. However, deletion of DST1, the gene encoding the yeast homolog of TFIIS, had only a small effect on transcriptional fidelity, as determined by this assay. In contrast, strains containing a deletion of RPB9, which encodes a small core subunit of Pol II, were found to engage in error-prone transcription. rpb9Delta strains also had increased steady-state levels of can1-100 mRNA, consistent with transcriptional errors that decrease the normal sensitivity of the can1-100 transcript to nonsense-mediated decay, a pathway that degrades mRNAs with premature stop codons. Sequences of cDNAs from rpb9Delta strains confirmed a significantly increased occurrence of transcriptional substitutions and insertions. These results suggest that Rpb9 plays an important role in maintaining transcriptional fidelity, whereas TFIIS may serve a different primary purpose.

Synthesis and biological evaluation of two chemically modified peptide epitopes for the class I MHC protein HLA-B*2705

The T-cell receptor of a CD8(+) T-cell recognises peptide epitopes bound by class I major histocompatibility complex (MHC) glycoproteins presented in a groove on their upper surface. Within the groove of the MHC molecule are 6 pockets, two of which mostly display a high degree of specificity for binding amino acids capable of making conserved and energetically favourable contacts with the MHC. One type of MHC molecule, HLA-B*2705, preferentially binds peptides containing an arginine at position 2. In an effort to increase the affinity of peptides for HLA-B*2705, potentially leading to better immune responses to such a peptide, we synthesised two modified epitopes where the amino acid at position 2 involved in anchoring the peptide to the class I molecule was replaced with the alpha-methylated beta,gamma-unsaturated arginine analogue 2-(S)-amino-5-guanidino-2-methyl-pent-3-enoic acid. The latter was prepared via a multi-step synthetic sequence, starting from alpha-methyl serine, and incorporated into dipeptides which were fragment-coupled to resin-bound heptameric peptides yielding the target nonameric sequences. Biological characterisation indicated that the modified peptides were poorer than the native peptides at stabilising empty class I MHC complexes, and cells sensitised with these peptides were not recognised as well by cognate CD8(+) T-cells, where available, compared to those sensitised with the native peptide. We suggest that the modifications made to the peptide have decreased its ability to bind to the peptide binding groove of HLA-B*2705 molecules which may explain the decrease in recognition by cytotoxic T-cells when compared to the native peptide.

Kinetic Analysis of Pseudomonas aeruginosa Arginine Deiminase Mutants and Alternate Substrates Provides Insight into Structural Determinants of Function

L-Arginine deiminase from Pseudomonas aeruginosa (PaADI) catalyzes the hydrolysis of arginine to citrulline and ammonia. PaADI belongs to the guanidino group-modifying enzyme superfamily (GMSF), which conserves backbone fold and a Cys-, His-, and Asp-based catalytic core. In this paper the contributions made by the PaADI core residues Cys406, His278, and Asp166 and the contribution from the neighboring Asp280 (conserved in most but not all GMSF members) to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were accessed by kinetic analysis of site-directed mutants. In addition, solution hydrolysis in a chemical model of the S-alkylthiouronium intermediate was examined to reveal the importance of general base catalysis in the enzymatic reaction. Substitutions of the active site gating residue Arg401, the l-arginine C(alpha)NH(3)(+)(COO(-)) binding residues, Arg185, Arg243, and Asn160, or the His278 hydrogen bond partner, Glu224, were found to cause dramatic reductions in the enzyme turnover rate. These results are interpreted to suggest that electrostatic interactions play a dominant role in PaADI catalysis. Structural variations observed in P. aeruginosa GMSF enzymes PaADI, agmatine deiminase (PaAgDI), and N(omega),N(omega)-dimethylarginine dimethylaminohydrolase (PaDDAH) indicate an early divergence of the encoding genes. Arginine analogues that are known substrates for PaAgDI and PaDDAH were tested with PaADI to define clear boundaries of biochemical function in the three hydrolases. The conservation of a catalytic core associated with the common chemical function and the divergence of substrate-binding residues (as well as one key catalytic residue) to expand the substrate range provide insight into the evolution of the catalysts that form the GMSF.